Interaction of 14-3-3 with Bid during seizure-induced neuronal death

Seizure-induced neuronal death may involve coordinated intracellular trafficking and protein-protein interactions of members of the Bcl-2 family. The 14-3-3 proteins are known to sequester certain pro-apoptotic members of this family. BH3-interacting domain death agonist (Bid) may contribute to seizure-induced neuronal death, although regulation by 14-3-3 has not been reported. In this study we examined whether 14-3-3 proteins interact with Bid during seizure-induced neuronal death. Brief seizures were evoked in rats by intraamygdala microinjection of kainic acid to elicit unilateral hippocampal CA3 neuronal death. Coimmunoprecipitation analysis demonstrated that although Bcl-2-associated death promoter (Bad) constitutively bound 14-3-3, there was no interaction between Bid and 14-3-3 in control brain. Seizures triggered Bid cleavage and a commensurate increase in binding of Bid to 14-3-3 within injured hippocampus. Casein kinases I and II, which can inactivate Bid by phosphoserine/threonine modification, did not coimmunoprecipitate with Bid. The largely uninjured contralateral hippocampus did not exhibit Bid cleavage or binding of 14-3-3 to Bid. In vitro experiments confirmed that 14-3-3beta is capable of binding truncated Bid, likely in the absence of phosphoserine/threonine modification. These data suggest 14-3-3 proteins may target active as well as inactive conformations of pro-apoptotic Bcl-2 death agonists, highlighting novel targets for intervention in seizure-induced neuronal death.     

Linking Aβ42-Induced Hyperexcitability to Neurodegeneration,Learning and Motor Deficits,and a Shorter Lifespan in an Alzheimer’s Model

Alzheimer’s disease (AD) is the most prevalent form of dementia in the elderly. β-amyloid (Aβ) accumulation in the brain is thought to be a primary event leading to eventual cognitive and motor dysfunction in AD. Aβ has been shown to promote neuronal hyperactivity, which is consistent with enhanced seizure activity in mouse models and AD patients. Little, however, is known about whether, and how, increased excitability contributes to downstream pathologies of AD. Here, we show that overexpression of human Aβ42 in a Drosophila model indeed induces increased neuronal activity. We found that the underlying mechanism involves the selective degradation of the A-type K+ channel, Kv4. An age-dependent loss of Kv4 leads to an increased probability of AP firing. Interestingly, we find that loss of Kv4 alone results in learning and locomotion defects, as well as a shortened lifespan. To test whether the Aβ42-induced increase in neuronal excitability contributes to, or exacerbates, downstream pathologies, we transgenically over-expressed Kv4 to near wild-type levels in Aβ42-expressing animals. We show that restoration of Kv4 attenuated age-dependent learning and locomotor deficits, slowed the onset of neurodegeneration, and partially rescued premature death seen in Aβ42-expressing animals. We conclude that Aβ42-induced hyperactivity plays a critical role in the age-dependent cognitive and motor decline of this Aβ42-Drosophila model, and possibly in AD.     

Shal/K(v)4 channels are required for maintaining excitability during repetitive firing and normal locomotion in Drosophila

Background

Rhythmic behaviors, such as walking and breathing, involve the coordinated activity of central pattern generators in the CNS, sensory feedback from the PNS, to motoneuron output to muscles. Unraveling the intrinsic electrical properties of these cellular components is essential to understanding this coordinated activity. Here, we examine the significance of the transient A-type K⁺ current (I_A), encoded by the highly conserved Shal/K_v4 gene, in neuronal firing patterns and repetitive behaviors. While I_A is present in nearly all neurons across species, elimination of I_A has been complicated in mammals because of multiple genes underlying I_A, and/or electrical remodeling that occurs in response to affecting one gene.

Methodology/Principal Findings

In Drosophila, the single Shal/K_v4 gene encodes the predominant I_A current in many neuronal cell bodies. Using a transgenically expressed dominant-negative subunit (DNK_v4), we show that I_A is completely eliminated from cell bodies, with no effect on other currents. Most notably, DNK_v4 neurons display multiple defects during prolonged stimuli. DNK_v4 neurons display shortened latency to firing, a lower threshold for repetitive firing, and a progressive decrement in AP amplitude to an adapted state. We record from identified motoneurons and show that Shal/K_v4 channels are similarly required for maintaining excitability during repetitive firing. We then examine larval crawling, and adult climbing and grooming, all behaviors that rely on repetitive firing. We show that all are defective in the absence of Shal/K_v4 function. Further, knock-out of Shal/K_v4 function specifically in motoneurons significantly affects the locomotion behaviors tested.

Conclusions/Significance

Based on our results, Shal/K_v4 channels regulate the initiation of firing, enable neurons to continuously fire throughout a prolonged stimulus, and also influence firing frequency. This study shows that Shal/K_v4 channels play a key role in repetitively firing neurons during prolonged input/output, and suggests that their function and regulation are important for rhythmic behaviors.     

Elektronenmikroskopische Untersuchungen über die Spermatogenese

Zusammenfassung Die Struktur und Genese der oligopyrenen Spermien von Melania libertina Gould wurden licht- und elektronenmikroskopisch untersucht. Die atypischen Spermatiden enthalten außer den bekannten Organellen des Cytoplasmas sehr viele elektronendichte Granula, die innerhalb der erweiterten Zisternen des endoplasmatischen Retikulums zum Vorschein kommen. Ihre Größe und Form schwanken beträchtlich; die rundlichen Granula haben Durchmesser von 20 bis 80 m. Die Granula verschmelzen zu großen, lichtmikroskopisch sichtbaren Körperchen, die RNA-Reaktion geben. Es wurde nachgewiesen, daß die Ribonucleoproteine in partikulärer Form durch die Kernporen in das Cytoplasma und weiter in die Zisternen des endoplasmatischen Retikulums übertreten. Damit ist die Annahme berechtigt, daß die in Zisternen auftretenden Körperchen keine Viren sind, sondern die aus dem Kern in die Zisternen übergetretenen RNP-Komplexe.Eine chemische Umwandlung der RNP in PAS-reaktionpositive Substanz wurde lichtmikroskopisch nachgewiesen.Im Karyoplasma des fertigen, oligopyrenen Spermatozoons ist eine fädige Struktur zu erkennen.Die Untersuchungen wurden durchgeführt mit Unterstützung durch The United States Public Health Service Grant-RG-8327.     

Tenascin-C enhances crosstalk signaling of integrin αvβ3/PDGFR-β complex by SRC recruitment promoting PDGF-induced proliferation and migration in smooth muscle cells

Migration and proliferation of smooth muscle cells (SMCs) are key events during neointimal formation in pathological conditions of vessels. Tenascin-C (TNC) is upregulated in the developing neointima of lesions. We evaluated the effects of TNC on responses of SMCs against platelet-derived growth factor (PDGF) stimulation. TNC coated on substrate promoted PDGF-BB-induced proliferation and migration of rat SMC cell line A10 in BrdU incorporation and transwell assays, respectively. Immunoblotting showed that TNC substrate enhanced autophosphorylation of PDGFR-β after PDGF-BB stimulation. Integrin αvβ3 is known to be a receptor for TNC in SMCs. In immunofluorescence and immunoblot of integrin αv subunit, clustering of αv-positive focal adhesions and upregulated αv expression were observed in the cells on TNC substrate. Immunoprecipitation using anti-integrin αvβ3 antibody demonstrated that PDGFR-β and integrin αvβ3 were co-precipitated and that the relative amount of PDGFR-β after the stimulation was increased by TNC treatment. TNC also promoted phosphorylation of focal adhesion kinase (FAK) at tyrosine (Y) 397 and Y925. The phosphorylated FAK was localized at focal adhesions in immunofluorescence. Phosphorylated SRC at Y418 was also seen at focal adhesions. Immunoprecipitation with αv antibody showed increased SRC association with the integrin signaling complex in the cells on TNC after PDGF treatment. In the cells on TNC substrate, crosstalk signaling between integrin αvβ3 and PDGFR-β could be amplified by SRC and FAK recruited to focal adhesions, followed by enhanced proliferation and migration of A10 cells by PDGF-BB.